Bacterial and fungal gut microbiota of supralittoral talitrid amphipods feeding on brown macroalgae and paper

Some macroalgae drift on the ocean and are stranded on coasts, and these stranded brown macroalgae are regarded to be degraded by organisms. Alginate is a major component of brown macroalgae. An uncovering of how carbon is cycled through brown macroalgae is needed to deeply understand coastal ecosystems. In this study, to gain insights into metabolism of brown macroalgae and alginate in the organisms, we initially confirmed that supralittoral talitrid amphipods (beach fleas or sandhoppers collected on the Shibagaki coast in Ishikawa Prefecture, Japan) fed on the brown macroalgae. We then isolated bacteria such as Vibrio sp. with alginate-assimilating capability from the gut of the amphipods. Metagenomic analysis of the gut of amphipods housed in several conditions (e.g. macroalgae or paper as feed, non-sterilized or sterilized environment) showed no condition-dependent compositions of bacteria and fungi, but Vibrio sp. were detected at high frequency, in good agreement with the isolation of Vibrio sp. An intervention study using antibiotics showed that amphipods fed on algae or paper at about the same rate in the presence or absence of antibiotics, and that the antibiotics had no effects on the life span. Moreover, intervention with antibiotics completely killed Vibrio sp. and some other bacteria, and had significant effects on the composition of the flora in the gut, with elimination of the variations observed in the guts of amphipods housed without antibiotics. These data suggest that microbes that were killed by antibiotics, including Vibrio sp., in the gut of talitrid amphipods are not essential for assimilation of brown macroalgae.


Introduction
Brown macroalgae have important roles in coastal ecosystems and are also promising carbon sources for biorefinery [1][2][3]. It has been estimated that about 173 TgC yr -1 of macroalgal carbon could be sequestered [4]. Alginate is one of the major carbon sources in brown macroalgae, with levels of up to 33.5% [1]. An uncovering of how carbon is cycled through brown macroalgae is needed to deeply understand coastal ecosystems, and this requires knowledge of how brown macroalgae, and especially alginate, are degraded, metabolized, and recycled by organisms. Some macroalgae drift on the ocean and are stranded on coasts, and these stranded brown macroalgae are degraded by microorganisms [5] and by talitrid amphipods (beach fleas and sandhoppers) [6,7]. Talitrid amphipods are typical organisms in the supralittorial zone and are key components of the sandy beach food web [8]. These amphipods are found on the coasts in Japan, especially under brown macroalgae [9]. With regard to bacteria, Vibrio sp. have been identified as decomposers of alginate and producers of alginate lyases [10,11]. Abdelrhman et al. suggested involvement of gut microbiota in carbon source assimilation in talitrid amphipods [8].
To understand further how brown macroalgae, and especially alginate, are degraded and metabolized by talitrid amphipods, in the current study we first confirmed that talitrid amphipods fed on stranded brown macroalgae, which indicates that talitrid amphipods degrade and metabolize the brown macroalgae in their guts. To understand the mechanism of degradation and metabolism of the brown macroalgae, we focused on microbes in the amphipod gut, initially by isolating alginate-assimilating bacteria, and then analyzing microbiota in the gut of amphipods housed under several conditions with or without antibiotics.

Feeding experiments
Talitrid amphipods (beach hoppers), sand and brown macroalgae (gulfweed) that had been stranded on the beach were collected at a sandy beach (a non-prohibited area) on the Shibagaki coast (36˚57' 08.9" N, 136˚45' 47.2" E) in Ishikawa Prefecture, Japan, on July 3, 2018 and Oct. 3,2018. Beach hoppers were housed in a tank (W 27 cm × D 17 cm × H 17 cm) containing sand on which the brown macroalgae or paper (Kimwiper, Wiper S-200, Nippon Paper Crecia, Tokyo, Japan) were placed. To avoid the amphipods hopping out, the tank was covered with a net, in addition to a lid. Similar tanks without beach hoppers were used as negative controls. The tanks were kept in a room at 25˚C. All macroalgae and sand used in the study were collected on the Shibagaki coast.
Guts were isolated from talitrid amphipods that had fed on brown macroalgae, using a sterilized tweezer under a microscope (Leica, EZ4D). The gut was suspended in 1.0 mL of sterilized 0.9% NaCl and the suspension (100 μL) was directly spread on solid NTA or NYTA medium. For the enrichment culture, the suspension (100 μL) was inoculated into 3.0 mL liquid NTA or NYTA medium, aerobically cultivated for 3-5 days, and diluted 100-and 10,000-fold in sterilized 0.9% NaCl. The diluted solution was spread on solid NTA or NYTA medium. Colonies were purified using the same solid medium. The alginate-degrading activity of the isolated strain was visualized with Gram's iodine (Sigma, HT902) [12] or TLC [13]. Mixtures of DEH and oligoalginates were prepared through digestion of sodium alginate by exotype (Atu3025) and endo-type (A1-I) alginate lyase [14]. For the Gram's iodine method, strains were cultivated in NTA liquid medium for 2 days, washed with 3.0% NaCl, and adjusted to OD 600 of 1.0 with 3.0% NaCl. This suspension (5.0 μL) was spotted onto NTA solid medium, further incubated for 3 days, and stained with Gram's iodine. For TLC, strains were cultivated in NTA liquid medium containing 2.0% sodium alginate for appropriate periods. The supernatant (5.0 μL) of the culture was spotted on HPLC Silica gel 60F254 (Merck), developed with solvent (1-butanol/acetate/water = 3:2:2), and visualized by heating after spraying with 10% sulfuric acid in ethanol.
The primers used in the study are listed in Table 1. To identify the isolated bacteria, 16S rRNA was amplified with colony PCR using primers 27F and 1492R and KOD FX Neo (Toyobo, Osaka, Japan), purified, and sequenced with each of primers 27F and 1492R. Sequence data were analyzed using EzBiocloud [15].
To measure growth (OD 600 ) of the isolated bacteria, the bacteria were precultured in 3.0 mL of NTA or NYTA liquid medium, collected by centrifugation, washed twice with 3.0% NaCl, and suspended in 3.0% NaCl to reach OD 600 of 0.10. The suspension (50 μL) was inoculated into 3.0 mL liquid medium and cultivated.

First metagenomic analysis
Each stranded brown macroalgae (gulfweed) was considered to be a separate residence of the talitrid amphipods. Collection was performed on Oct. 17, 2019 on the Shibagaki coast. Talitrid amphipods under the macroalgae, the sand around the macroalgae, and the macroalgae itself were collected from two separate residence sites, which are referred to as brown macroalgae α and β (S1A Fig in S1 File). Amphipods from each residence were housed with the macroalgae or paper as above in a 1 L beaker containing 300 mL of sand. The beaker was covered with aluminum foil. For sterilized conditions, beakers containing sand and either algae or paper were autoclaved. The algae were dried at 60˚C overnight before autoclaving. The experimental conditions were as shown in Table 2. The amphipods were washed twice in 5.0 mL of sterilized 3.0% (w/v) NaCl by vortex and 10 washed amphipods were released into each beaker. Talitrid amphipods that had fed on macroalgae or paper and had been housed for 7 or 14 days were observed by microscopy to identify their sex and species [9,16] and were then sterilized in 70% ethanol for about 30 sec. The gut was isolated from each individual and metagenomic DNA was purified from the gut using a FastDNA Spin Kit For Soil (MP Biomedicals). Metagenomic (amplicon) analysis was conducted at Fasmac (Atsugi, Japan) as follows. The quality and quantity of the DNA was checked with Qubit (Thermo Fisher Scientific). 16S rRNA V3-V4, ITS2, and 18S rRNA V9 regions were amplified with 1st PCR using ExTaq HS (Takara, Otsu, Japan) and primers 341F/805R [17], gITS7/ITS4 [18], and 1389F/1510R [19], respectively (Table 1). 2nd PCR was conducted with ExTaq HS using the purified product of 1st PCR as a template and primers 2nd Forward and 2nd Reverse (Table 1) [20]. DADA2 was used to process the data [21]. Taxonomy was assigned to the 16S or 18S data using the SILVA reference database (silva_132_release) [22] from which the V3-V4 or V9 region was extracted using the 341F/805R or 1389F/1510R primer information. A Naive Bayes model was trained using the extracted regions. Taxonomy was assigned to the ITS2 data using the UNITE database [23]. A Naive Bayes model was trained using the database without extraction.

Intervention study and second metagenomic analysis
Stock solutions of the antibiotics ampicillin, streptomycin, and kanamycin were prepared in water at 50, 50, and 25 mg/mL, respectively, and sterilized with a filter (pore size 0.2 μm). To determine the concentrations of antibiotics for the study, suspensions (OD 600 of 1.0) of P. homiensis SK6416 and V. chemaguriensis SK6422 (Table 3) were prepared as above, diluted to OD 600 of 0.1 and 0.01, and spotted onto solid NTA medium containing the antibiotics. The solid media were incubated for 4 days and stained with Gram's iodine.
Talitrid amphipods, sand and stranded brown macroalgae were collected on June 6, 2020 and Sep. 24, 2020. The amphipods were housed as described above, but with the following modifications. The sand and brown macroalgae were dried at 110˚C and 60˚C overnight, respectively. The 1 L beaker containing dried sand (300 mL) was sterilized by autoclaving. The dried brown macroalgae or paper were also separately autoclaved. The sterilized algae or sterilized paper was placed onto the sterilized beakers and sand, and then sterilized water without or with antibiotics (0.45 mg/mL streptomycin, 0.45 mg/mL ampicillin, and 0.225 mg/mL kanamycin) was added to a level of 1.0 mm above the sand surface. Ten amphipods washed with sterilized water without or with antibiotics were released into each beaker and housed for 14 days for the second metagenomic analysis ( Table 2). This analysis was conducted as above. At 0, 3 and 7 days, photos were taken to observe the effects on feeding.
For isolation of microbes from the guts of talitrid amphipods housed with algae and antibiotics (condition I, Table 2), the guts were isolated and suspended in 3.0% NaCl, as described above. The suspension was spread on NTA, NYTA, or Marine broth 2216 (Difco) solid media in the presence or absence of antibiotics directly or after enrichment cultivation in the same liquid medium. When colonies appeared on solid media, the colony was purified using the same medium.
For life span experiments, talitrid amphipods, sand and stranded brown macroalgae were collected on April 14, 2021, Aug. 31, 2021, and Oct. 8,2021. Nine amphipods washed with sterilized water and nine washed with sterilized water containing antibiotics were released into

Feeding experiments
The Shibagaki coast is a sandy beach located in Ishikawa Prefecture, Japan, facing the Japan Sea. Many brown macroalgae (gulfweed) were stranded on the beach (S1A Fig in S1 File), and d ns: not specified. "α" and "β" indicate stranded macroalgae α and β from which amphipods were collected (S1A Fig in S1 File). e Condition A: Non-sterilized stranded macroalgae β.  many beach hoppers (talitrid amphipods) were observed under these macroalgae (S1B Fig in  S1 File). The mature males of the talitrid amphipods were identified as Platorchestia joi, Sinorchestia nipponensis, and Sinorchestia sinensis ( Table 2). Some immature amphipods (juveniles) were regarded as male due to immature morphological features ( Table 2). All other amphipods were regarded as female, but their genera and species could not be identified due to the absence of distinguishable morphological features (Table 2). Nanjo previously suggested that talitridae found under stranded brown macroalgae might feed on the algae [24], but it has not been shown experimentally that amphipods in the Talitridae family feed on brown macroalgae. Crawley and Hyndes found that Allorchestes compressa (a species of amphipod in the Dogielinotidae family) exhibits a clear preference for brown macroalgae over red algae and seagrass as food [6]. To determine if talitrid amphipods feed on stranded brown macroalgae, the talitrid amphipods were released into tanks containing brown macroalgae or paper as feed. The talitrid amphipods fed on both brown macroalgae and paper (Fig 1). Thus, amphipods in the Talitridae family appear to assimilate brown macroalgae, as well as paper, through their guts.

Isolation of alginate-assimilating bacteria from the gut of talitrid amphipods
To address the role of the gut in assimilation of brown macroalgae, we attempted to isolate gut microbes with the capability to assimilate alginate, the main carbohydrate of brown macroalgae [1]. As shown in Table 3, 10 strains were isolated as alginate-assimilating bacteria and were identified as Paracoccus fistulariae, Paracoccus homiensis, Vibrio algivorus, Vibrio chemaguriensis, and Vibrio neocaledonicus, in agreement with identification of Vibrio sp. as decomposers of alginate and producers of alginate lyases [10,11]. The isolated bacteria exhibited alginate-dependent growth (no growth in NT containing no organic carbon, but growth in NTA [NT plus 0.5% alginate]) and a growth defect in the absence of 3.0% NaCl (Fig 2), as well as showing alginate-degrading activity (S2 Fig in S1 File). V. algivorus SA2(T) strain has been identified as an agarose-and alginate-assimilating bacterium [25] and the genes responsible for decomposition, transport, and metabolism of alginate have been identified [26]. Doi et al. found that V. algivorus SA2(T) grows in the presence of 1-14% NaCl, but not in the absence of NaCl [25], while the optimal growth condition for V. neocaledonicus requires salinity of 50 g/L [27]. These findings are consistent with the properties of the bacteria isolated in this study.

Metagenomic analysis of the gut of talitrid amphipods
Next, we examined the gut microbiota of talitrid amphipods feeding on brown macroalgae. The effects of feed (brown macroalgae or paper), sterilization (non-sterilized or sterilized environment), period of housing (7 or 14 days), residence on the sandy beach (brown macroalgae α or β), sex (male or female), and each individual on the microbiota were evaluated. Amphipods were housed under conditions B-G shown in Taxonomy was assigned to the 16S (Fig 3), ITS (Fig 4), and 18S data (S3 Fig in S1 File). In the 18S data, Metazoa (Animalia) comprised the vast majority of all samples from amphipods (Nos. 2 to 35) (S3 Fig in S1 File). Accordingly, representative 18S sequences displayed similarity to each other and a Blast search showed that one of the sequences had strong similarity (99% identity, 164/165 sequences) to the partial sequence of 18S ribosomal RNA genes of A. angusta (ID: JX545346.1) and the genera Hyalella (e.g. MT823241.1), both of which belong to the order Amphioda. Thus, we concluded that almost all of the 18S data corresponded to the talitrid amphipods from which DNA was extracted, and we stopped further analysis of these data.
In contrast to the 18S data, bar plots of the relative frequency of bacterial taxa (16S data) and fungal taxa (ITS2 data) showed variations (Figs 3 and 4). In alpha-and beta-diversity analyses using the 16S data, there was no difference shown by the Shannon alpha-diversity metric (S4A Fig in S1 File) and beta diversity analysis using weighted UniFrac distances showed no clustering of samples ( S5A Fig in S1 File). In bar plots of the 16S data, Vibrio was the major genus and was found in all 35 samples (obtained from conditions A-G) at frequencies of 0.1-95.5% (Fig 3, S1 Table). This distribution of the genus Vibrio was in good accord with that Vibrio sp. were isolated from guts of amphipods and proven to assimilate alginate (Fig 2; S2 Fig in  S1 File). The genus Vibrio was found at low frequency (0.6%) in stranded brown macroalgae (sample no. 1, condition A), but at higher frequency in the gut of the amphipods (S1 Table). This suggests that the amphipod gut was a more suitable environment than the brown macroalgae for Vibrio. Vibrio sp. have also been detected in hadal amphipods [28].
The distribution of the genus Vibrio was not dependent on the feed (algae or paper) and other conditions (Fig 3, S1 Table). The genus Paracoccus, which was also isolated from guts of amphipods and proven to assimilate alginate (Fig 2; S2 Fig in S1 File), was also distributed, but at much lower frequencies (0.1-0.8%) in fewer samples (Nos. 6, 9, 10, 12, 18, 32, 33, and 34) (S1 Table). The genera Pseudoalteromonas and Albimonas were distributed (Fig 3, S1 Table). Pseudoalteromonas sp. have been reported to have alginate utilization genes [10,29] and Pseudoalteromonas sp. in the gut of amphipods may assimilate alginate. Candidatus Hepatoplasma was also clearly distributed (Fig 3, S1 Table), but only at high frequency in 3 samples [Nos. 2 (91.2%), 25 (95.2%), and 28 (85.1%)]. This distribution of Candidatus Hepatoplasma is in accord with that detected in hadal amphipods [28]. The genus Thioclava was only found at high frequency in the stranded brown macroalgae. Collectively, condition-dependent compositions of bacteria were not observed.
Alpha-and beta-diversity analyses were conducted using the ITS2 data. Data from condition E differed from those from condition D in the Shannon alpha-diversity metric (S4B Fig in  S1 File). Beta diversity analysis using weighted UniFrac distances for ITS2 data indicated that only data from condition B clustered separately from data from condition E (S5B Fig in   Fig 3. Bar plot of the relative frequency of 16S bacterial data at the genus level. The frequencies of some genera are shown in S1 Table. https://doi.org/10.1371/journal.pone.0279834.g003  Table. https://doi.org/10.1371/journal.pone.0279834.g004 S1 File). In bar plots, the genus Cyberlindnera was clearly detected in all samples except 13,14,27,30, and 31 (Fig 4, S2 Table). Unidentified genera were also observed, especially in samples 2-5, 14-17, 24, and 26 (Unidentified � 3) and in samples 2, 13, and 14 (Unidentified � 2) (Fig 4, S2 Table).

Effects of antibiotics
The metagenomic analysis above revealed the composition of the genera of bacteria and fungi, but no condition (i.e., feed, sterilization, or period)-dependent composition was observed. To obtain more information on the relationship between feed (algae or paper) and microbial flora, in particular for the alginate-assimilating bacteria such as Vibrio and Paracoccus, in the gut of talitrid amphipods, an intervention study using antibiotics was conducted.
Treatment with 0.45 mg/mL streptomycin, 0.45 mg/mL ampicillin, and 0.225 mg/mL kanamycin was first shown to exhibit antibiotic effects against the alginate-assimilating bacteria (P. homiensis SK6416 and V. chemaguriensis SK6422; Table 3) isolated in this study (S6 Fig in S1 File). Then, talitrid amphipods (Nos. 37-45, Table 2) were housed in the presence of the same antibiotic concentrations with algae (condition I) or paper (condition J) under sterilized conditions for 14 days ( Table 2, Figs 3 and 4). As a control, the amphipods were housed with algae (No. 36, condition H, Table 2) in the absence of antibiotics under sterilized conditions for 14 days. The effects of the residence on the sandy beach, sex, and each individual on the microbiota were not included in this experiment, since these effects were unclear in the first metagenomic study described above.
We anticipated that talitrid amphipods feeding on algae would die or their feeding on algae would be slowed after the gut bacteria were killed by the antibiotics, if these bacteria were essential for assimilation of algae. However, the amphipods fed on algae or paper at about the same rate, irrespective of the presence or absence of antibiotics (S7 Fig in S1 File). To examine the effect of antibiotics on life span, the amphipods were housed with or without antibiotics for a long period. A count of the number of surviving amphipods indicated no effect of the antibiotics on the life span (Fig 5).
To identify the microbes in the gut of amphipods housed in sterilized condition with or without antibiotics, DNA was isolated from the guts of amphipods housed for 14 days (1 amphipod from condition H [algae as feed, without antibiotics], 5 from condition I [algae as feed, with antibiotics], and 4 from condition J [paper as feed, with antibiotics]) and metagenomic analysis was conducted using 16S and ITS2 data obtained from these samples. As shown in Figs 3 and 4 (conditions H-J), both 16S and ITS2 data showed that intervention with antibiotics completely killed Vibrio sp. and had significant effects on the composition of the flora in the gut, with elimination of the variation in the guts of amphipods housed without antibiotics.
For 16S data, antibiotics with algae as feed had a composition consisting of unassigned bacteria, and genera Echinicola and Pseudomonas, while antibiotics with paper as feed gave a composition of genera Rhodococcus, Ochrobactrum, and Pseudomonas (Fig 3 and S1 Table). We note that these genera were also sometimes detected in the absence of antibiotics, although at very low frequency, indicating that these genera flourished after antibiotic killing of bacteria such as Vibrio sp., Paracoccus sp., and Pseudoalteromonas sp., which had prospered in the absence of antibiotics.
For ITS2 data, antibiotics with algae as feed gave a composition of genera Acremonium  Table). Beta diversity analysis using weighted UniFrac distances for 16S and ITS2 data indicated that data from conditions H, I, and J clustered separately from each other (S8 Fig in  S1 File). The antibiotics seemed not to affect the fungi directly, which suggests that antibiotics have indirect effects on the fungi by causing significant changes in the gut bacterial composition.
We attempted to isolate microbes from guts of talitrid amphipods housed with algae and antibiotics (condition I, Table 2). Although Echinicola and Pseudomonas are major genera in the guts of these amphipods (Fig 3, S1 Table), we failed to isolate these genera. Instead, we isolated two bacteria using marine broth, which were identified as Aeromonas bivalvium and Stenotrophomonas maltophilia (Table 4). A. bivalvium was not detected in metagenomic analysis, while S. maltophilia was detected at higher frequency in the presence of antibiotics compared to that in the absence of antibiotics (S1 Table). Alginate-assimilation by these two bacteria were not confirmed due to the absence of growth in NTA or TA medium (S9A Fig in S1 File). The alginate-degrading activity of the two bacteria was also unclear: both bacteria showed a halo in Gram's iodine staining, but alginate degradation was not found in TLC analysis (S9B Fig in S1 File).

Role of Vibrio sp. in the gut of talitrid amphipods
Animal glycoside hydrolase 7 (GH7) cellobiohydrolase in the marine wood borer Limnoria quadripunctata (LqCel7B) has recently been characterized and shown to be secreted in the gut of L. quadripunctata for wood degradation [30]. A Blastp search using the primary structure of LqCel7B gave three homologs in amphipods, including the talitrid amphipod Trinorchestia longiramus (S3 Table), suggesting that GH7 cellobiohydrolase functions in the gut of talitrid amphipods. This enzyme may digest brown macroalgae for assimilation and to enhance access of microbes, especially Vibrio sp., to alginate.
Microbes in the gut of talitrid amphipods may have effects on the hosts. Abdelrhman et al. [8] detected glycosyl hydrolase family 48 gene, which is a measure of cellulolytic bacterial diversity in environmental samples [31], in the gut microbiota of five species of supralittoral talitrid amphipods, suggesting involvement of gut microbiota in carbon source assimilation. In the current study, Vibrio sp. was detected at high frequency in the gut, and killing of bacteria such as Vibrio sp., Paracoccus sp., and Pseudoalteromonas sp. with antibiotics had no effect on the life span of amphipods (Fig 5). These results suggest that microbes that were killed by antibiotics, including Vibrio sp. and Pseudoalteromonas sp., are not essential for assimilation of brown macroalgae in these amphipods. The possibility remains that microbes that flourished after antibiotic killing of bacteria such as Vibrio sp. may have roles in assimilation of brown macroalgae.